Solid dressing for treating wounded tissue

ABSTRACT

Disclosed are solid dressings for treated wounded tissue in mammalian patients, such as a human, comprising a haemostatic layer consisting essentially of a fibrinogen component and thrombin, wherein the thrombin is present in an amount between 0.250 Units/mg of fibrinogen component and 0.062 Units/mg of fibrinogen component. Also disclosed are methods for treating wounded tissue.

FIELD OF THE INVENTION

The present application is a continuation of U.S. patent applicationSer. No. 11/882,879, entitled, “Solid Dressing for Treating WoundedTissue,” filed Aug. 6, 2007, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a solid dressing for treating woundedtissue in a mammalian patient, such as a human. The materials andmethods available to stop bleeding in pre-hospital care (gauzedressings, direct pressure, and tourniquets) have, unfortunately, notchanged significantly in the past 2000 years. See L. Zimmerman et al.,Great Ideas in the History of Surgery (San Francisco, Calif NormanPublishing; 1993), 31. Even in trained hands they are not uniformlyeffective, and the occurrence of excessive bleeding or fatal hemorrhagefrom an accessible site is not uncommon. See J. M. Rocko et al., J.Trauma 22:635 (1982).

Mortality data from Vietnam indicates that 10% of combat deaths were dueto uncontrolled extremity hemorrhage. See SAS/STAT Users Guide, 4th ed.(Cary, N.C.: SAS Institute Inc; 1990). Up to one third of the deathsfrom ex-sanguination during the Vietnam War could have been prevented bythe use of effective field hemorrhage control methods. See SAS/STATUsers Guide, 4th ed. (Cary, N.C.: SAS Institute Inc; 1990).

Although civilian trauma mortality statistics do not provide exactnumbers for pre-hospital deaths from extremity hemorrhage, case andanecdotal reports indicate similar occurrences. See J. M. Rocko et al..These data suggest that a substantial increase in survival can beaffected by the pre-hospital use of a simple and effective method ofhemorrhage control.

There are now in use a number of newer haemostatic agents that have beendeveloped to overcome the deficiencies of traditional gauze bandages.These haemostatic agents include the following:

-   -   Microporous polysaccharide particles (TraumaDEX®, Medafor Inc.,        Minneapolis, Minn.);    -   Zeolite (QuikClot®, Z-Medica Corp, Wallington, Conn.);    -   Acetylated poly-N-acetyl glucosamine (Rapid Deployment Hemostat™        (RDH), Marine Polymer Technologies, Danvers, Mass.);    -   Chitosan (HemCon® bandage, HemCon Medical Technologies inc.,        Portland Oreg.);    -   Liquid Fibrin Sealants (Tisseel V H, Baxter, Deerfield, Ill.)    -   Human fibrinogen and thrombin on equine collagen (TachoComb-S,        Hafslund Nycomed Pharma, Linz, Austria);    -   Microdispersed oxidized cellulose (m•doc™, Alltracel Group,        Dublin, Ireland);    -   Propyl gallate (Hemostatin™, Analytical Control Systems Inc.,        Fishers, Ind.);    -   Epsilon aminocaproic acid and thrombin (Hemarrest™ patch,        Clarion Pharmaceuticals, Inc);    -   Purified bovine corium collagen (Avitene® sheets (non-woven web        or Avitene Microfibrillar Collagen Hemostat (MCH), Davol, Inc.,        Cranston, R.I.);    -   Controlled oxidation of regenerated cellulose (Surgicel®,        Ethicon Inc., Somerville, N.J.);    -   Aluminum sulfate with an ethyl cellulose coating (Sorbastace        Microcaps, Hemostace, LLC, New Orleans, La.);    -   Microporous hydrogel-forming polyacrylamide (BioHemostat,        Hemodyne, Inc., Richmond Va.); and    -   Recombinant activated factor VII (NovoSeven®, NovoNordisk Inc.,        Princeton, N.J.). These agents have met with varying degrees of        success when used in animal models of traumatic injuries and/or        in the field.

One such agent is a starch-based haemostatic agent sold under the tradename TraumaDEX™. This product comprises microporous polysaccharideparticles that are poured directly into or onto a wound. The particlesappear to exert their haemostatic effect by absorbing water from theblood and plasma in the wound, resulting in the accumulation andconcentration of clotting factors and platelets. In two studies of alethal groin wound model, however, this agent showed no meaningfulbenefit over standard gauze dressings. See McManus et al., BusinessBriefing: Emergency Medical Review 2005, pp. 76-79 (presently availableon-line at www.touchbriefings.com/pdf/1334/Wedmore.pdf).

Another particle-based agent is QuickClot™ powder, a zeolite granularhaemostatic agent that is poured directly into or onto a wound. Thezeolite particles also appear to exert their haemostatic effect throughfluid absorption, which cause the accumulation and concentration ofclotting factors and platelets. Although this agent has been usedsuccessfully in some animal studies, there remains concern about theexothermic process of fluid absorption by the particles. Some studieshave shown this reaction to produce temperatures in excess of 143° C. invitro and in excess of 50° C. in vivo, which is severe enough to causethird-degree burns. See McManus et al., Business Briefing: EmergencyMedical Review 2005, at 77. The exothermic reaction of QuikClot™ hasalso been observed to result in gross and histological tissue changes ofunknown clinical significance. Acheson et al., J. Trauma 59:865-874(2005).

Unlike these particle-based agents, the Rapid Deployment Hemostat™appears to exert its haemostatic effect through red blood cellaggregation, platelet activation, clotting cascade activation and localvasoconstriction. The Rapid Deployment Hemostat™ is an algae-deriveddressing composed of poly-N-acetyl-glucosamine. While the originaldressing design was effective in reducing minor bleeding, it wasnecessary to add gauze backing in order to reduce blood loss in swinemodels of aortic and liver injury. See McManus et al., BusinessBriefing: Emergency Medical Review 2005, at 78.

Another poly-N-acetyl-glucosamine-derived dressing is the HemConTMChitosan Bandage, which is a freeze-dried chitosan dressing purportedlydesigned to optimize the mucoadhesive surface density and structuralintegrity of the chitosan at the site of the wound. The HemCon™ ChitosanBandage apparently exerts its haemostatic effects primarily throughadhesion to the wound, although there is evidence suggesting it may alsoenhance platelet function and incorporate red blood cells into the clotit forms on the wound. This bandage has shown improved hemostasis andreduced blood loss in several animal models of arterial hemorrhage, buta marked variability was observed between bandages, including thefailure of some due to inadequate adherence to the wound. See McManus etal., Business Briefing: Emergency Medical Review 2005, at 79.

Liquid fibrin sealants, such as Tisseel VH, have been used for years asan operating room adjunct for hemorrhage control. See J. L. Garza etal., J. Trauma 30:512-513 (1990); H. B. Kram et al., J. Trauma30:97-101(1990); M. G. Ochsner et al., J. Trauma 30:884-887 (1990); T.L. Matthew et al., Ann. Thorac. Surg. 50:40-44 (1990); H. Jakob et al.,J. Vasc. Surg., 1:171-180 (1984). The first mention of tissue glue usedfor hemostasis dates back to 1909. See Current Trends in Surgical TissueAdhesives: Proceedings of the First International Symposium on SurgicalAdhesives, M. J. MacPhee et al., eds. (Lancaster, Pa.: TechnomicPublishing Co; 1995). Liquid fibrin sealants are typically composed offibrinogen and thrombin, but may also contain Factor XIII/XIIIa, eitheras a by-product of fibrinogen purification or as an added ingredient (incertain applications, it is therefore not necessary that FactorXIII/Factor XIIIa be present in the fibrin sealant because there issufficient Factor XIII/XIIIa, or other transaminase, endogenouslypresent to induce fibrin formation). As liquids, however, these fibrinsealants have not proved useful for treating traumatic injuries in thefield.

Dry fibrinogen-thrombin dressings having a collagen support (e.g.TachoComb™, TachoComb™ H and TachoSil available from Hafslund NycomedPharma, Linz, Austria) are also available for operating room use in manyEuropean countries. See U. Schiele et al., Clin. Materials 9:169-177(1992). While these fibrinogen-thrombin dressings do not require thepre-mixing needed by liquid fibrin sealants, their utility for fieldapplications is limited by a requirement for storage at 4° C. and thenecessity for pre-wetting with saline solution prior to application tothe wound. These dressings are also not effective against high pressure,high volume bleeding. See Sondeen et al., J. Trauma 54:280-285 (2003).

A dry fibrinogen/thrombin dressing for treating wounded tissue is alsoavailable from the American Red Cross (ARC). As disclosed in U.S. Pat.No. 6,762,336, this particular dressing is composed of a backingmaterial and a plurality of layers, the outer two of which containfibrinogen (but no thrombin) while the inner layer contains thrombin andcalcium chloride (but no fibrinogen). While this dressing has showngreat success in several animal models of hemorrhage, the bandage isfragile, inflexible, and has a tendency to break apart when handled. SeeMcManus et al., Business Briefing: Emergency Medical Review 2005, at78.; Kheirabadi et al., J. Trauma 59:25-35 (2005).

Other fibrinogen/thrombin-based dressings have also been proposed. Forexample, U.S. Pat. No. 4,683,142 discloses a resorptive sheet materialfor closing and healing wounds which consists of a glycoprotein matrix,such as collagen, containing coagulation proteins, such as fibrinogenand thrombin. U.S. Pat. No. 5,702,715 discloses a reinforced biologicalsealant composed of separate layers of fibrinogen and thrombin, at leastone of which also contains a reinforcement filler such as PEG, PVP, BSA,mannitol, FICOLL, dextran, myo-inositol or sodium chlorate. U.S. Pat.No. 6,056,970 discloses dressings composed of a bioabsorbable polymer,such as hyaluronic acid or carboxymethylcellulose, and a haemostaticcomposition composed of powdered thrombin and/or powdered fibrinogen.U.S. Pat. No. 7,189,410 discloses a bandage composed of a backingmaterial having thereon: (i) particles of fibrinogen; (ii) particles ofthrombin; and (iii) calcium chloride. U.S. Patent ApplicationPublication No. US 2006/0155234 Al discloses a dressing composed of abacking material and a plurality of fibrinogen layers which havediscrete areas of thrombin between them. To date, none of thesedressings have been approved for use or are available commercially.

Accordingly, there remains a need in the art for a solid dressing thatcan be used to treat wounded tissue, particularly wounded tissueresulting from traumatic injury in the field.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a soliddressing that can treat wounded mammalian tissue, particularly woundedtissue resulting from a traumatic injury. It is further an object of thepresent invention to provide a method of treating wounded mammaliantissue, particularly human tissue. Other objects, features andadvantages of the present invention will be set forth in the detaileddescription of preferred embodiments that follows, and will in part beapparent from that description and/or may be learned by practice of thepresent invention. These objects and advantages will be realized andattained by the compositions and methods described in this specificationand particularly pointed out in the claims that follow.

In accordance with these and other objects, a first embodiment of thepresent invention is direct to a solid dressing for treating woundedtissue in a mammal comprising at least one haemostatic layer consistingessentially of a fibrinogen component and thrombin, wherein the thrombinis present in an amount between about 0.250 Units/mg of fibrinogencomponent and 0.062 Units/mg of fibrinogen component.

Another embodiment is directed to a method of treating wounded tissueusing a solid dressing comprising at least one haemostatic layerconsisting essentially of a fibrinogen component and thrombin, whereinthe thrombin is present in an amount between about 0.250 Units/mg offibrinogen component and 0.062 Units/mg of fibrinogen component.

Other embodiments are directed to similar solid dressings wherein theamount of thrombin is between 0.125 Units/mg of fibrinogen component and0.080 Units/mg of fibrinogen component., and the use of the same fortreating wounded tissue.

It is to be understood that the foregoing general description and thefollowing detailed description of preferred embodiments are exemplaryand explanatory only and are intended to provide further explanation,but not limitation, of the invention as claimed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A and FIG. 1B are graphs depicting the results of the EVPA andAdherence Assays.

FIG. 2 is a diagram of the set-up for the EVPA Assay.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsmentioned herein are incorporated by reference.

As used herein, use of a singular article such as “a,” “an,” and “the”is not intended to excluded pluralities of the article's object unlessthe context clearly and unambiguously dictates otherwise.

“Patient” as used herein refers to human or animal individuals in needof medical care and/or treatment.

“Wound” as used herein refers to any damage to any tissue of a patientwhich results in the loss of blood from the circulatory system and/orany other fluid from the patient's body. The tissue may be an internaltissue, such as an organ or blood vessel, or an external tissue, such asthe skin. The loss of blood may be internal, such as from a rupturedorgan, or external, such as from a laceration. A wound may be in a softtissue, such as an organ, or in hard tissue, such as bone. The damagemay have been caused by any agent or source, including traumatic injury,infection or surgical intervention.

“Resorbable material” as used herein refers to a material that is brokendown spontaneously in and/or by the mammalian body into components whichare consumed or eliminated in such a manner as not to interferesignificantly with wound healing and/or tissue regeneration, and withoutcausing any significant metabolic disturbance.

“Stability” as used herein refers to the retention of thosecharacteristics of a material that determine activity and/or function.

“Suitable” as used herein is intended to mean that a material does notadversely affect the stability of the dressings or any componentthereof.

“Binding agent” as used herein refers to a compound or mixture ofcompounds that improves the adherence and/or cohesion of the componentsof the haemostatic layer(s) of the dressings.

“Solubilizing agent” as used herein refers to a compound or mixture ofcompounds that improves the dissolution of a protein or proteins inaqueous solvent.

“Filler” as used herein refers to a compound or mixture of compoundsthat provide bulk and/or porosity to the haemostatic layer(s) of adressing.

“Release agent” as used herein refers to a compound or mixture ofcompounds that facilitates removal of a dressing from a manufacturingmold.

“Foaming agent” as used herein refers to a compound or mixture ofcompounds that produces gas when hydrated under suitable conditions.

“Solid” as used herein is intended to mean that the dressing will notsubstantially change in shape or form when placed on a rigid surface,wound-facing side down, and then left to stand at room temperature for24 hours.

A first preferred embodiment of the present invention is directed to asolid dressing for treating wounded tissue in a patient which comprisesa haemostatic layer consisting essentially of a fibrinogen component andthrombin, wherein the thrombin is present in an amount between 0.250Units/mg of fibrinogen component and 0.062 Units/mg of fibrinogencomponent.

As used herein, “consisting essentially of” is intended to mean that thefibrinogen component and the thrombin are the only necessary andessential ingredients of the haemostatic layer(s) of the solid dressingwhen it is used as intended to treat wounded tissue. Accordingly, thehaemostatic layer may contain other ingredients in addition to thefibrinogen component and the thrombin as desired for a particularapplication, but these other ingredients are not required for the soliddressing to function as intended under normal conditions, i.e. theseother ingredients are not necessary for the fibrinogen component andthrombin to react and form enough fibrin to reduce the flow of bloodand/or fluid from normal wounded tissue when that dressing is applied tothat tissue under the intended conditions of use. If, however, theconditions of use in a particular situation are not normal, for examplethe patient is a hemophiliac suffering from Factor XIII deficiency, thenthe appropriate additional components, such as Factor XIII/XIIIa or someother transaminase, may be added to the haemostatic layer(s) withoutdeviating from the spirit of the present invention. Similarly, the soliddressing of the present invention may contain one or more of thesehaemostatic layers as well as one or more other layers, such as one ormore support layers (e.g. a backing material or an internal supportmaterial) and release layers.

Other preferred embodiments are directed to similar solid dressingswherein the amount of thrombin is between 0.125 Units/mg of fibrinogencomponent and 0.080 Units/mg of fibrinogen component. Still otherpreferred embodiments of the present invention are directed to similarsolid dressings wherein the amount of thrombin is (all values being±0.0009): 0.250 Units/mg of fibrinogen component; 0.125 Units/mg offibrinogen component; 0.100 Units/mg of fibrinogen component; 0.080Units/mg of fibrinogen component; 0.062 Units/mg of fibrinogencomponent; 0.050 Units/mg of fibrinogen component; and 0.025 Units/mg offibrinogen component.

Another preferred embodiment of the present invention is directed to amethod for treating wounded tissue in a mammal, comprising placing asolid dressing of the present invention to wounded tissue and applyingsufficient pressure to the dressing for a sufficient time for enoughfibrin to form to reduce the loss of blood and/or other fluid from thewound.

According to certain embodiments of the present invention, thehaemostatic layer(s) of the solid dressing is formed or cast as a singlepiece. According to certain other embodiments of the present invention,the haemostatic layer is made or formed into or from a single source,e.g. an aqueous solution containing a mixture of the fibrinogencomponent and the thrombin. With each of these embodiments of thepresent invention, the haemostatic layer(s) is preferably substantiallyhomogeneous throughout.

According to other preferred embodiments, the haemostatic layer(s) ofthe solid dressing are composed of a plurality of particles, each ofwhich consists essentially of a fibrinogen component and thrombin.According to such embodiments, the haemostatic layer may also contain abinding agent to facilitate or improve the adherence of the particles toone another and/or to any support layer(s). Illustrative examples ofsuitable binding agents include, but are not limited to, sucrose,mannitol, sorbitol, gelatin, maltose, hyaluron and its derivatives, suchas hyaluronic acid, povidone, starch, chitosan and its derivatives (e.g.NOCC-chitosan), and cellulose derivatives, such ascarboxymethylcellulose, as well as mixtures of two or more thereof.

The haemostatic layer(s) of the solid dressing may also optionallycontain one or more suitable fillers, such as sucrose, lactose, maltose,silk, fibrin, collagen, albumin, polysorbate (Tween™), chitin, chitosanand its derivatives, such as NOCC-chitosan, alginic acid and saltsthereof, hyaluron and its derivatives, such as hyaluronic acid,cellulose and derivatives thereof, proteoglycans, glycolic acidpolymers, lactic acid polymers, glycolic acid/lactic acid co-polymers,and mixtures of two or more thereof.

The haemostatic layer of the solid dressing may also optionally containone or more suitable solubilizing agents, such as sucrose, dextrose,mannose, trehalose, mannitol, sorbitol, hyaluron and its derivatives,such as hyaluronic acid, albumin, sorbate, polysorbate (Tween™),sorbitan (SPAN™) and mixtures of two or more thereof.

The haemostatic layer of the solid dressing may also optionally containone or more suitable foaming agents, such as a mixture of aphysiologically acceptable acid (e.g. citric acid or acetic acid) and aphysiologically suitable base (e.g. sodium bicarbonate or calciumcarbonate). Other suitable foaming agents include, but are not limitedto, dry particles containing pressurized gas, such as sugar particlescontaining carbon dioxide (see, e.g., U.S. Pat. No. 3,012,893) or otherphysiologically acceptable gases (e.g. Nitrogen or Argon), andpharmacologically acceptable peroxides.

The haemostatic layer(s) of the solid dressing may also optionallycontain a suitable source of calcium ions, such as calcium chloride,and/or a fibrin cross-linker, such as a transaminase (e.g. FactorXIII/XIIIa) or glutaraldehyde.

The haemostatic layer of the solid dressing is preferably prepared bymixing aqueous solutions of the fibrinogen component and the thrombinunder conditions which minimize the activation of the fibrinogencomponent by the thrombin. The mixture of aqueous solutions is thensubjected to a process such as lyophilization or freeze-drying to reducethe moisture content to the desired level, i.e. to a level where thedressing is solid and therefore will not substantially change in shapeor form upon standing, wound-facing surface down, at room temperaturefor 24 hours. Similar processes that achieve the same result, such asdrying, spray-drying, vacuum drying and vitrification, may also beemployed.

As used herein, “moisture content” refers to the amount freely-availablewater in the dressing. “Freely-available” means the water is not boundto or complexed with one or more of the non-liquid components of thedressing. The moisture content referenced herein refers to levelsdetermined by procedures substantially similar to the FDA-approved,modified Karl Fischer method (Meyer and Boyd, Analytical Chem.,31:215-219, 1959; May et al., J. Biol. Standardization, 10:249-259,1982; Centers for Biologics Evaluation and Research, FDA, Docket No.89D-0140, 83-93; 1990) or by near infrared spectroscopy. Suitablemoisture content(s) for a particular solid dressing may be determinedempirically by one skilled in the art depending upon the intendedapplication(s) thereof.

For example, in certain embodiments of the present invention, highermoisture contents are associated with more flexible solid dressings.Thus, in solid dressings intended for extremity wounds, it may bepreferred to have a moisture content of at least 6% and even morepreferably in the range of 6% to 44%.

Similarly, in other embodiments of the present invention, lower moisturecontents are associated with more rigid solid dressings. Thus, in soliddressings intended for flat wounds, such as wounds to the abdomen orchest, it may be preferred to have a moisture content of less than 6%and even more preferably in the range of 1% to 6%.

Accordingly, illustrative examples of suitable moisture contents forsolid dressings include, but are not limited to, the following (eachvalue being ±0.9%): less than 53%; less than 44%; less than 28%; lessthan 24%; less than 16%; less than 12%; less than 6%; less than 5%; lessthan 4%; less than 3%; less than 2.5%; less than 2%; less than 1.4%;between 0 and 12%, non-inclusive; between 0 and 6%; between 0 and 4%;between 0 and 3%; between 0 and 2%; between 0 and 1%; between 1 and 16%;between 1 and 11%; between 1 and 8%; between 1 and 6%; between 1 and 4%;between 1 and 3%; between 1 and 2%; and between 2 and 4%.

The fibrinogen component in the haemostatic layer(s) of the soliddressings may be any suitable fibrinogen known and available to thoseskilled in the art. The fibrinogen component may also be a functionalderivative or metabolite of a fibrinogen, such the fibrinogen α, βand/or γ chains, soluble fibrin I or fibrin II, or a mixture of two ormore thereof. A specific fibrinogen (or functional derivative ormetabolite) for a particular application may be selected empirically byone skilled in the art. As used herein, the term “fibrinogen” isintended to include mixtures of fibrinogen and small mounts of FactorXIII/Factor XIIIa, or some other such transaminase. Such small amountsare generally recognized by those skilled in the art as usually beingfound in mammalian fibrinogen after it has been purified according tothe methods and techniques presently known and available in the art andtypically range from 0.1 to 20 Units/mL

Preferably, the fibrinogen employed as the fibrinogen component of thesolid dressing is a purified fibrinogen suitable for introduction into amammal Typically, such fibrinogen is a part of a mixture of human plasmaproteins which include Factor XIII/XIIIa and have been purified to anappropriate level and virally inactivated. A preferred aqueous solutionof fibrinogen for preparation of a solid dressing contains around 37.5mg/mL fibrinogen at a pH of around 7.4±0.1, although a pH in the rangeof 5.5-8.5 may be acceptable. Suitable fibrinogen for use as thefibrinogen component has been described in the art, e.g. U.S. Pat. No.5,716,645, and similar materials are commercially available, e.g. fromsources such as Sigma-Aldrich, Enzyme Research Laboratories,Haematologic Technologies and Aniara.

Preferably, the fibrinogen component is present in an amount of fromabout 1.5 to about 13.0 mg (±0.9 mg) of fibrinogen per square centimeterof solid dressing, and more preferably from about 3.0 to about 13.0mg/cm². Greater or lesser amounts, however, may be employed dependingupon the particular application intended for the solid dressing. Forexample, according to certain embodiments where increased adherence isdesired, the fibrinogen component is present in an amount of from about11.0 to about 13.0 mg (±0.9 mg) of fibrinogen per square centimeter ofsolid dressing. Likewise, according to certain embodiments which areintended for treating low pressure-containing vessels, lower levels ofthe fibrinogen component may be employed.

While any suitable mammalian thrombin may be used in a solid dressing,the thrombin employed in the haemostatic layer is preferably alyophilized mixture of human plasma proteins which has been sufficientlypurified and virally inactivated for the intended use of the soliddressing. Suitable thrombin is available commercially from sources suchas Sigma-Aldrich, Enzyme Research Laboratories, HaematologicTechnologies and Biomol International. A particularly preferred aqueoussolution of thrombin for preparing a solid dressing contains thrombin ata potency of between 10 and 2000±50 International Units/mL, and morepreferred at a potency of 25+2.5 International Units/mL Otherconstituents may include albumin (generally about 0.1 mg/mL) and glycine(generally about 100 mM±0.1 mM). The pH of this particularly preferredaqueous solution of thrombin is generally in the range of 6.5-7.8, andpreferably 7.4±0.1, although a pH in the range of 5.5-8.5 may beacceptable.

In addition to the haemostatic layer(s), the solid dressing mayoptionally further comprise one or more support layers. As used herein,a “support layer” refers to a material that sustains or improves thestructural integrity of the solid dressing and/or the fibrin clot formedwhen such a dressing is applied to wounded tissue.

According to certain preferred embodiments of the present invention thesupport layer comprises a backing material on the side of the dressingopposite the side to be applied to wounded tissue. Such a backingmaterial may be affixed with a physiologically-acceptable adhesive ormay be self-adhering (e.g. by having a sufficient surface staticcharge). The backing material may comprise one or more resorbablematerials or one or more non-resorbable materials or mixtures thereof.Preferably, the backing material is a single resorbable material

Any suitable resorbable material known and available to those skilled inthe art may be employed in the present invention. For example, theresorbable material may be a proteinaceous substance, such as silk,fibrin, keratin, collagen and/or gelatin. Alternatively, the resorbablematerial may be a carbohydrate substance, such as alginates, chitin,cellulose, proteoglycans (e.g. poly-N-acetyl glucosamine), hyaluron andits derivatives, such as hyaluronic acid, glycolic acid polymers, lacticacid polymers, or glycolic acid/lactic acid co-polymers. The resorbablematerial may also comprise a mixture of proteinaceous substances or amixture of carbohydrate substances or a mixture of both proteinaceoussubstances and carbohydrate substances. Specific resorbable material(s)may be selected empirically by those skilled in the art depending uponthe intended use of the solid dressing.

According to certain preferred embodiments of the present invention, theresorbable material is a carbohydrate substance. Illustrative examplesof particularly preferred resorbable materials include, but are notlimited to, the materials sold under the trade names VICRYL™ (a glycolicacid/lactic acid copolymer) and DEXON™ (a glycolic acid polymer).

Any suitable non-resorbable material known and available to thoseskilled in the art may be employed as the backing material. Illustrativeexamples of suitable non-resorbable materials include, but are notlimited to, plastics, silicone polymers, gauze, latexes, paper and paperproducts, and the like.

According to other preferred embodiments, the support layer comprises aninternal support material Such an internal support material ispreferably fully embedded or contained within a haemostatic layer of thesolid dressing., although it may be placed between two adjacenthaemostatic layers in certain embodiments. As with the backing material,the internal support material may be a resorbable material or anon-resorbable material, or a mixture thereof, such as a mixture of twoor more resorbable materials or a mixture of two or more non-resorbablematerials or a mixture of resorbable material(s) and non-resorbablematerial(s).

According to still other preferred embodiments, the support layer maycomprise a front support material on the wound-facing side of thedressing, i.e. the side to be applied to wounded tissue. As with thebacking material and the internal support material, the front supportmaterial may be a resorbable material or a non-resorbable material, or amixture thereof, such as a mixture of two or more resorbable materialsor a mixture of two or more non-resorbable materials or a mixture ofresorbable material(s) and non-resorbable material(s).

According to still other preferred embodiments, the solid dressingcomprises both a backing material and an internal support material inaddition to the haemostatic layer(s), i.e. the solid dressing comprisestwo support layers in addition to the haemostatic layer(s). According tostill other preferred embodiments, the solid dressing comprises both afront support material and an internal support material in addition tothe haemostatic layer(s). According to still other preferredembodiments, the solid dressing comprises a backing material, a frontsupport material and an internal support material in addition to thehaemostatic layer(s).

According to certain embodiments of the present invention, particularlywhere the dry dressing is manufactured using a mold, the dry dressingsmay also optionally further comprise a release layer in addition to thehemostatic layer(s) and support layer(s). As used herein, a “releaselayer” refers to a layer containing one or more agents (“releaseagents”) which promote or facilitate removal of the dry dressing from amold in which it has been manufactured. A preferred such agent issucrose, but other suitable release agents include gelatin, mannitol,sorbitol, polysorbate (Tween™), sorbitan (SPAN™), lactose, maltose,trehalose, hyaluron and its derivatives, such as hyaluronic acid,sorbate, glucose and mixtures of two or more thereof. Alternatively,such one or more release agents may be contained in the hemostaticlayer.

The various layers of the inventive dressings may be affixed to oneanother by any suitable means known and available to those skilled inthe art. For example, a physiologically-acceptable adhesive may beapplied to a backing material (when present), and the haemostaticlayer(s) subsequently affixed thereto.

In certain embodiments of the present invention, thephysiologically-acceptable adhesive has a shear strength and/orstructure such that the backing material can be separated from thefibrin clot formed by the haemostatic layer after application of thedressing to wounded tissue. In other embodiments, thephysiologically-acceptable adhesive has a shear strength and/orstructure such that the backing material cannot be separated from thefibrin clot after application of the bandage to wounded tissue.

Suitable fibrinogen components and suitable thrombin for the haemostaticlayer(s) of the solid dressing may be obtained from any appropriatesource known and available to those skilled in the art, including, butnot limited to, the following: from commercial vendors, such asSigma-Aldrich and Enzyme Research Laboratories; by extraction andpurification from human or mammalian plasma by any of the methods knownand available to those skilled in the art; from supernatants or pastesderived from plasma or recombinant tissue culture, viruses, yeast,bacteria, or the like that contain a gene that expresses a human ormammalian plasma protein which has been introduced according to standardrecombinant DNA techniques; and/or from the fluids (e.g. blood, milk,lymph, urine or the like) of transgenic mammals (e.g. goats, sheep,cows) that contain a gene which has been introduced according tostandard transgenic techniques and that expresses the desired fibrinogencomponent and/or desired thrombin.

According to certain preferred embodiments of the present invention, thefibrinogen component is a mammalian fibrinogen such as bovinefibrinogen, porcine fibrinogen, ovine fibrinogen, equine fibrinogen,caprine fibrinogen, feline fibrinogen, canine fibrinogen, murinefibrinogen or human fibrinogen. According to other embodiments, thefibrinogen component is bird fibrinogen or fish fibrinogen. According tostill other embodiments, the fibrinogen component is human fibrinogen,human fibrinogen a chain, human fibrinogen β chain, human fibrinogen γchain, human fibrin I, human fibrin II, or a mixture of two or morethereof. According to any of these embodiments, the fibrinogen may berecombinantly produced fibrinogen or transgenic fibrinogen. As notedabove, the fibrinogen may also contain small amounts (e.g. _-_% of totalprotein) of a transaminase, such as Factor XIII/XIIIa.

According to certain preferred embodiments of the present invention, thethrombin is a mammalian thrombin, such as bovine thrombin, porcinethrombin, ovine thrombin, equine thrombin, caprine thrombin, felinethrombin, canine thrombin, murine thrombin and human thrombin. Accordingto other embodiments, the thrombin is bird thrombin or fish thrombin.According to any of these embodiments, the thrombin may be recombinantlyproduced thrombin or transgenic thrombin.

As a general proposition, the purity of the fibrinogen component and/orthe thrombin for use in the solid dressing will be a purity known to oneof ordinary skill in the relevant art to lead to the optimal efficacyand stability of the protein(s). Preferably, the fibrinogen componentand/or the thrombin has been subjected to multiple purification steps,such as precipitation, concentration, diafiltration and affinitychromatography (preferably immunoaffinity chromatography), to removesubstances which cause fragmentation, activation and/or degradation ofthe fibrinogen component and/or the thrombin during manufacture, storageand/or use of the solid dressing. Illustrative examples of suchsubstances that are preferably removed by purification include: proteincontaminants, such as inter-alpha trypsin inhibitor and pre-alphatrypsin inhibitor; non-protein contaminants, such as lipids; andmixtures of protein and non-protein contaminants, such as lipoproteins.

The amount of the fibrinogen component employed in the solid dressing ispreferably selected to optimize both the efficacy and stability thereof.As such, a suitable concentration for a particular application of thesolid dressing may be determined empirically by one skilled in therelevant art. According to certain preferred embodiments of the presentinvention, when the fibrinogen component is human fibrinogen, the amountof fibrinogen employed is between 1.5 mg and 13.0 mg (each ±0.9 mg) persquare centimeter of solid dressing, more preferably between 3.0 mg and13.0 mg per square centimeter and most preferably between 11.0 mg and13.0 mg per square centimeter.

During use of the solid dressing, the fibrinogen component and thethrombin are preferably activated at the time the dressing is applied tothe wounded tissue by the endogenous fluids of the patient escaping fromthe hemorrhaging wound. Alternatively, in situations where fluid lossfrom the wounded tissue is insufficient to provide adequate hydration ofthe protein layers, the fibrinogen component and/or the thrombin may beactivated by a suitable, physiologically-acceptable liquid, optionallycontaining any necessary co-factors and/or enzymes, prior to or duringapplication of the dressing to the wounded tissue.

In some embodiments of the present invention, the haemostatic layer(s)may also contain one or more supplements, such as growth factors, drugs,polyclonal and monoclonal antibodies and other compounds. Illustrativeexamples of such supplements include, but are not limited to, thefollowing: fibrinolysis inhibitors, such as aprotonin, tranexamic acidand epsilon-amino-caproic acid; antibiotics, such as tetracycline andciprofloxacin, amoxicillin, and metronidazole; anticoagulants, such asactivated protein C, heparin, prostacyclins, prostaglandins(particularly (PGI₂), leukotrienes, antithrombin III, ADPase, andplasminogen activator; steroids, such as dexamethasone, inhibitors ofprostacyclin, prostaglandins, leukotrienes and/or kinins to inhibitinflammation; cardiovascular drugs, such as calcium channel blockers,vasodilators and vasoconstrictors; chemoattractants; local anestheticssuch as bupivacaine; and antiproliferative/antitumor drugs such as5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such asgangcyclovir, zidovudine, amantidine, vidarabine, ribaravin,trifluridine, acyclovir, dideoxyuridine and antibodies to, viralcomponents or gene products; cytokines, such as alpha- or beta- orgamma-Interferon, alpha- or beta-tumor necrosis factor, andinterleukins; colony stimulating factors; erythropoietin; antifungals,such as diflucan, ketaconizole and nystatin; antiparasitic gents, suchas pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsinand alpha-1-antichymotrypsin; anesthetics, such as bupivacaine;analgesics; antiseptics; hormones; vitamins and other nutritionalsupplements; glycoproteins; fibronectin; peptides and proteins;carbohydrates (both simple and/or complex); proteoglycans;antiangiogenins; antigens; lipids or liposomes; oligonucleotides (senseand/or antisense DNA and/or RNA); and gene therapy reagents. In otherembodiments of the present invention, the backing layer and/or theinternal support layer, if present, may contain one or more supplements.According to certain preferred embodiments of the present invention, thetherapeutic supplement is present in an amount greater than itssolubility limit in fibrin.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

EXAMPLES

The ability of the dressings to seal an injured blood vessel wasdetermined by an ex vivo porcine arteriotomy (EVPA) performance test,which was first described in U.S. Pat. No. 6,762,336. The EVPAperformance test evaluates the ability of a dressing to stop fluid flowthrough a hole in a porcine artery. While the procedure described inU.S. Pat. No. 6,762,336 has been shown to be useful for evaluatinghaemostatic dressings, it failed to replicate faithfully therequirements for success in vivo. More specifically, the proceduredisclosed in U.S. Pat. No. 6,762,336 required testing at 37° C.,whereas, in the real world, wounds are typically cooler than that. Thisdecreased temperature can significantly reduce the rate of fibrinformation and its haemostatic efficacy in trauma victims. See, e.g.,Acheson et al., J. Trauma 59:865-874 (2005). The test in U.S. Pat. No.6,762,336 also failed to require a high degree of adherence of thedressing to the injured tissue. A failure mode in which fibrin forms butthe dressing fails to attach tightly to the tissue would, therefore, notbe detected by this test. Additionally, the pressure utilized in theprocedure (200 mHg) may be exceeded during therapy for some traumapatients. The overall result of this is that numerous animal tests,typically involving small animals (such as rats and rabbits), must beconducted to accurately predict dressing performance in large animal,realistic trauma studies and in the clinical environment.

In order to minimize the amount of time and the number of animal studiesrequired to develop the present invention, an improved ex vivo testingprocedure was developed. To accomplish this, the basic conditions underwhich the dressing test was conducted were changed, and the severity ofthe test parameters was increased to include testing at lowertemperatures (i.e. 29-33° C. vs. 37 ° C., representing the realphysiologic challenge at realistic wound temperatures (Acheson et al.,J. Trauma 59:865-874 (2005)), higher pressures (i.e. 250 mmHg vs. 200mmHg), a longer test period (3 minutes vs. 2 minutes) and larger sizedarterial injuries (U.S. Pat. No. 6,762,336 used an 18 gauge needlepuncture, whereas the revised procedure used puncture holes ranging from2.8 mm to 4 mm×6 mm)

In addition, a new test was derived to directly measure adherence of thedressing to the injured tissue. Both these tests showed greatly improvedstringency and are thus capable of surpassing the previous ex vivo testand replacing many in vivo tests for efficacy.

The following is a list of acronyms used in the Examples below:

-   CFB: Complete Fibrinogen Buffer (100 mM Sodium Chloride, 1.1 mM    Calcium Chloride, 10 mM Tris, 10 mM Sodium Citrate, 1.5% Sucrose,    Human Serum Albumin (80 mg/g of total protein) and Tween™ 80 (animal    source) 15 mg/g total protein)-   CTB: Complete Thrombin Buffer (150 mM Sodium Chloride, 40 mM Calcium    Chloride, 10 mM Tris and 100 mM L-Lysine with the addition of HSA at    100 ug/ml)-   ERL: Enzyme Research Laboratories-   EVPA: Ex Vivo Porcine Arteriotomy-   FD: Inventive haemostatic dressing-   HSA: Human Serum Albumin-   HD: A “sandwich” fibrin sealant haemostatic dressing as disclosed in    U.S. Pat. No. 6,762,336-   IFB: Incomplete Fibrinogen Buffer.; CFB without HSA and Tween-   PETG: Glycol-modified Polyethlylenetetrapthalate-   PPG: Polypropylene-   PVC: Poly vinyl chloride-   TRIS: trishydroxymethylaminomethane    (2-amino-2-hydroxymethyl-1,3-propanediol)

Example 1

Backing material (DEXONTM) was placed into 2.4×2.4 cm PETG molds.Twenty-five microliters of 2% sucrose was pipetted on top of each of thefour corners of the backing material. Once completed the molds wereplaced in a −80° C. freezer for at least 60 minutes.

Fibrinogen (Enzyme Research Laboratories™ (ERL) lot 3114) was formulatedin 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mMSodium Citrate, 1.5% Sucrose, Human Serum Albumin (HSA) at aconcentration of 80mg/g of total protein and 15 mg/g total protein ofTween™ 80 (animal source) (Complete Fibrinogen buffer (CFB)). Thefibrinogen concentration was adjusted to 37.5 mg/ml using CFB. The finalpH of the fibrinogen was 7.4±0.1. Once prepared the fibrinogen wasplaced on ice until use.

Thrombin was formulated in 150 mM Sodium Chloride, 40 mM CalciumChloride, 10 mM Tris and 100 mM L-Lysine with the addition of HSA at 100ug/ml (Complete Thrombin Buffer (CTB)). The fmal pH of the thrombin was7.4±0.1. The thrombin concentrations were adjusted with CFB to produce12.5 units/mg of Fibrinogen (upon mixing), which corresponded to 3120Units/ml thrombin prior to mixing Once prepared the thrombin was placedon ice until use.

The temperature of the fibrinogen and thrombin prior to dispensing was4° C.±2° C. Molds were removed from the -80° C. freezer and placed on acopper plate that was precooled on top of dry ice. A repeat pipettor wasfilled with fibrinogen and second repeat pipettor was filled withthrombin. Two ml of fibrinogen and 300 micro liters of thrombin weredispensed simultaneously into each mold. Once the molds were filled theywere returned to the -80° C. freezer for at least two hours before beingplaced into the freeze dryer. They were then lyophilized as describedbelow, and performance tested using the EVPA and Adherence Assays asdescribed below.

Example 2

Backing material was placed into each 1.5×1.5 cm PVC molds. Fifteenmicroliters of 2% sucrose was pipetted on top of each of the fourcorners of the backing material. A second piece of PETG plastic wasfitted on top of the 1.5×1.5 molds and held in place. This formed aclosed mold. The molds were then placed in a −80° C. freezer for atleast 60 minutes. Fibrinogen (ERL lot 3100) was formulated in CFB. Thefibrinogen concentration was adjusted to 37.5 mg/ml using CFB. The finalpH of the fibrinogen was 7.4±0.1. Once prepared the fibrinogen wasplaced on ice until use. Thrombin was formulated in CTB. The final pH ofthe thrombin was 7.4±0.1. The thrombin concentrations were adjustedusing CTB to deliver the following amounts 2.5, 0.25, 0.1, 0.05, 0.025,0.016, 0.0125 and 0.01 units/mg of Fibrinogen (upon mixing), whichcorresponded to 624, 62.4, 25, 12.5, 6.24, 3.99, 3.12, and 2.5 Units/mlthrombin prior to mixing Once prepared the thrombin was placed on iceuntil use. The temperature of the fibrinogen and thrombin prior todispensing was 4° C.±2° C. Molds were then removed from the −80° C.freezer and placed on an aluminum plate that was pre-cooled on top ofdry ice. Three holes were punched at the top of the mold using an 18gauge needle. One hole was used for injecting fibrinogen, the second forinjecting thrombin, and the third hole served as a vent to release airthat was displaced from inside the mold. A pipette was then filled withfibrinogen and a second pipette with thrombin. Simultaneously 0.78 ml offibrinogen and 0.17 ml of thrombin were injected via these pipettes intoeach mold. Once filled the molds were placed on top of a pool of liquidnitrogen for thirty seconds and then returned to the −80° C. freezer forat least two hours before being placed into the freeze dryer. They werethen lyophilized as described below, and performance tested using theEVPA and Adherence Assays as described below.

Example 3

Backing material was placed into 2.4×2.4 cm PVC molds. Twenty-fivemicroliters of 2% sucrose was pipetted on top of each of the fourcorners of the backing material. Once completed the molds were placed ina −80° C. freezer for at least 60 minutes. Fibrinogen (ERL lot 3100) wasformulated in CFB. The fibrinogen concentration was adjusted to 37.5mg/ml using CFB. The final pH of the fibrinogen was 7.4±0.1. Onceprepared the fibrinogen was placed on ice until use. Thrombin wasformulated in CTB. The final pH of the thrombin was 7.4±0.1. Using CTB,the thrombin concentrations were adjusted to deliver the followingamounts 0.125, 0.025, 0.0125, 0.00625 and 0.0031 units/mg of Fibrinogenupon mixing, which corresponded to 31.2, 6.24, 3.12, 1.56 and 0.78Units/ml thrombin prior to mixing. Once prepared the thrombin was placedon ice until use. The temperature of the fibrinogen and thrombin priorto dispensing was 4° C.±2° C. The molds were removed from the −80° C.freezer and placed on an aluminum plate that that was precooled on topof dry ice. A 3 ml syringe fitted with an 18 gauge needle was filledwith 2 ml of fibrinogen and a second, 1 ml, syringe fitted with a 22gauge needle was filled with 0.3 ml of thrombin. The contents of bothsyringes were dispensed simultaneously into each mold. Once filled themolds were placed on top of liquid nitrogen for thirty seconds and thenreturned to the −80° C. freezer for at least two hours before beingplaced into the freeze dryer. They were then lyophilized as describedbelow, and performance tested using the EVPA and Adherence Assays asdescribed below.

Example 4

Backing material was placed into PVC 2.4×2.4 cm molds. Twenty-fivemicroliters of 2% sucrose was pipetted on top of each of the fourcorners of the backing material. Once completed the molds were placed ina −80° C. freezer for at least 60 minutes. A vial containing 3 grams ofFibrinogen (Sigma™ Lot #F-3879) was removed the −20° C. freezer andplaced at 4° C. for 18 hours. The bottle was then removed from thefreezer and allowed to come to room temperature for 60 minutes. To thebottle, 60 ml of 37° C. water was added and allowed to mix for 15minutes at 37° C. Once in solution the fibrinogen was dialyzed againstincomplete fibrinogen buffer (IFB, which was CFB without HSA and Tween™)for 4 hours at room temperature. At the end of the four hours HSA wasadded to a concentration of 80 mg/g of total protein, and Tween™ 80(animal source) was added to a concentration of 15 mg/g total protein.The final pH of the fibrinogen was 7.4±0.1. The fibrinogen concentrationwas then adjusted to 37.5 mg/m with CFB. Once prepared the fibrinogenwas placed on ice until use. Thrombin was formulated in CTB. The finalpH of the thrombin was 7.4±0.1. Using CTB, the thrombin concentrationswere adjusted to deliver the following amounts 2.5, 0.25, 0.125, 0.083and 0.0625 units/mg of Fibrinogen (upon mixing) which corresponded to624, 62.4, 31.2, 20.8 and 15.6 Units/ml thrombin prior to mixing Onceprepared the thrombin was placed on ice until use. The temperature ofthe fibrinogen and thrombin prior to dispensing was 4° C.±2° C. Moldswere removed from the −80° C. freezer and placed on an aluminum platethat was that was precooled on top of dry ice. A 3 ml syringe fittedwith an 18 gauge needle was filled with 2 ml of fibrinogen and a second1 ml syringe fitted with a 22 gauge needle was filled with 0.3 ml ofthrombin. The contents of both syringes were dispensed simultaneouslyinto each mold. Once filled the molds were placed on top of liquidnitrogen for thirty seconds and then returned to the −80° C. freezer forat least two hours before being placed into the freeze dryer. They werethen lyophilized as described below, and performance tested using theEVPA and Adherence Assays as described below.

Example 5

Backing material was placed into 2.4×2.4 cm PVC molds. Twenty-fivemicroliters of 2% sucrose was pipetted on top of each of the fourcorners of the backing material. A second piece of PETG plastic was cutto fit on top of the molds and held in place by clips located at eachend of the mold, producing closed molds. Once completed the molds wereplaced in a −80° C. freezer for at least 60 minutes. Fibrinogen (ERL lot3060 was formulated in CFB. The final pH of the fibrinogen was 7.4±0.1.The fibrinogen concentration was adjusted to 37.5 mg/ml using CFB. Onceprepared the fibrinogen was placed on ice until use. Thrombin wasformulated in CTB. The final pH of the thrombin was 7.4±0.1. Using CTB,thrombin concentrations were adjusted to deliver the following amounts2.5, 0.25, 0.125, 0.083 and 0.062 units/mg of Fibrinogen (after mixing),which corresponded to 624, 62.4, 31.2, 20.8, and 15.6 Units/ml thrombin(prior to mixing). Once prepared the thrombin was placed on ice untiluse. The temperature of the fibrinogen and thrombin prior to dispensingwas 4° C.±2° C. Molds were removed from the −80° C. freezer and placedon an aluminum plate that was that was precooled on top of dry ice. A 3ml syringe fitted with an 18 gauge needle was filled with 2 ml offibrinogen and a second, 1 ml, syringe fitted with a 22 gauge needle wasfilled with 0.3 ml of thrombin. The contents of both syringes weredispensed simultaneously into each mold. Once filled the molds wereplaced on top of liquid nitrogen for thirty seconds and then returned tothe −80° C. freezer for at least two hours before being placed into thefreeze dryer. They were then lyophilized as described below, andperformance tested using the EVPA and Adherence Assays as describedbelow.

Example 6

Backing material was placed into 2.4×2.4 cm PVC molds. Twenty-fivemicroliters of 2% sucrose was pipetted on top of each of the fourcorners of the backing material. A second piece of PETG plastic was cutto fit on top of the 2.4×2.4 molds and held in place by the use of clipslocated at each end of the mold to create closed molds. The molds werethen placed in a −80° C. freezer for at least 60 minutes. A vialcontaining 3 grams of Fibrinogen (Sigma Lot #F-3879) was removed the−20° C. freezer and placed at 4° C. for 18 hours. The bottle was thenremoved from the freezer and allowed to come to room temperature for 60minutes. To the bottle, 60 ml of 37° C. water was added and allowed tomix for 15 minutes at 37° C. Once in solution the fibrinogen wasdialyzed against IFB. At the end of the four hours HSA was added to aconcentration of 80 mg/g of total protein, and Tween™ 80 (animal source)was added to a concentration of 15 mg/g total protein. The final pH ofthe fibrinogen was 7.4±0.1. The fibrinogen concentration was adjusted to37.5 mg/ml using CFB. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. Thrombin concentration was adjusted to deliver thefollowing amounts 2.5, 0.25, 0.125, 0.1 and 0.083 units/mg of Fibrinogen(upon mixing), which corresponded to 624, 62.4, 31.2, 24.96 and 20.79Units/ml thrombin (before mixing). Once prepared the thrombin was placedon ice until use. The temperature of the fibrinogen and thrombin priorto dispensing was 4° C.±2° C. Molds were removed from the −80° C.freezer and placed on an aluminum plate that was that was precooled ontop of dry ice. A 3 ml syringe fitted with an 18 gauge needle was filledwith 2 ml of fibrinogen and a second, 1 ml, syringe fitted with a 22gauge needle was filled with 0.3 ml of thrombin. The contents of bothsyringes were dispensed simultaneously into each mold. Once filled themolds were placed on top of liquid nitrogen for thirty seconds and thenreturned to the −80° C. freezer for at least two hours before beingplaced into the freeze dryer. They were then lyophilized as describedbelow, and performance tested using the EVPA and Adherence Assays asdescribed below.

Example 7

Backing material was placed into 2.4×2.4 cm PVC molds. Twenty-fivemicroliters of 2% sucrose was pipetted on top of each of the fourcorners of the backing material. A second piece of PETG plastic was cutto fit on top of the molds and held in place by the use of clips locatedat each end of the mold to create closed molds. Once completed, themolds were placed in a −80° C. freezer for at least 60 minutes.

A vial containing 3 grams of Fibrinogen (Sigma™ Lot# F-3879) was removedfrom the −20° C. freezer and placed at 4° C. for 18 hours. The bottlewas then allowed to come to room temperature for 60 minutes. To thebottle, 60 ml of 37° C. water was added and allowed to mix for 20minutes at 37° C. Once in solution, the fibrinogen was dialyzed againstIFB. At the end of the four hours, human serum albumin (HSA) was addedto a concentration of 80 mg/g of total protein, and Tween™ 80 (animalsource) was added to a concentration of 15 mg/g total protein. The finalpH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml using CFB. Once prepared the fibrinogen wasplaced on ice until use.

Thrombin was formulated in CTB. B. The final pH of the thrombin was7.4±0.1. Thrombin was adjusted to deliver the following amounts 2.5,0.25, 0.125, 0.08 and 0.06 units/mg of Fibrinogen (after mixing), whichcorresponded to 624, 62.4, 31.2, 20.8 and 15.6 Units/ml thrombin (priorto mixing) Once prepared the thrombin was placed on ice until use. Thetemperature of the fibrinogen and thrombin prior to dispensing was 4°C.±2° C. Molds were removed from the −80° C. freezer and placed on analuminum plate that was that was precooled on top of dry ice. A 3 mlsyringe fitted with an 18 gauge needle was filled with 2 ml offibrinogen and a second, lml, syringe fitted with a 22 gauge needle wasfilled with 0.3 ml of thrombin. The contents of both syringes weredispensed simultaneously into each mold. Once filled the molds wereplaced on top of liquid nitrogen for thirty seconds and then returned tothe −80° C. freezer for at least two hours before being placed into thefreeze dryer. They were then lyophilized as described below, andperformance tested using the EVPA and Adherence Assays as describedbelow.

Trilayer (Sandwich) Dressings

Trilayer dressings were produced using the process described in U.S.Pat. No. 6,762,336, using the same sources of fibrinogen and thrombin asutilized to produce the monolithic dressings above.

Results

The results of the EVPA and Adherence Assays are shown in FIGS. 1A and1B, respectively.

Conclusions:

Dressings produced with between 2.5 to 0.025 Thrombin Units/mg ofFibrinogen were active in both assays, while those with greater orlesser ratios of thrombin to fibrinogen were not. Significantly greateractivity was seen over the range of 2.5 to 0.05 Thrombin Units/mg ofFibrinogen. Greatly improved performance was seen between the ranges of0.25 to 0.062 Thrombin Units/mg of Fibrinogen, while optimum performancewas seen between the ranges of 0.125 to 0.08 Thrombin Units/mg ofFibrinogen. This contrasted with the dressings produced using theprocess described in U.S. Pat. No. 6,762,336 which reached fullperformance at 12.5 Thrombin Units/mg of Fibrinogen, with unacceptableperformance occurring as the thrombin concentration was diminished below12.5 Thrombin Units/mg of Fibrinogen, with essentially no activityremaining at 1.4 Thrombin Units/mg of Fibrinogen. This difference inboth the limits of performance and the optimum levels is all the moreprofound given that the performance of the Trilayer dressings from U.S.Pat. No. 6,762,336 was decreased by the use of decreasing amounts ofthrombin, while the dressing described herein showed an increasedactivity over this range.

Example 8

Monolithic dressings were manufactured as follows: backing material wascut and placed into each PETG 2.4×2.4 cm mold. Twenty-five microlitersof 2% sucrose was pipetted on top of each of the four corners of thebacking material. Once completed the molds were placed in a −80° C.freezer for at least 60 minutes.

For all dressings, ERL fibrinogen lot 3114 was formulated in CFB. Thefinal pH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. The thrombin was adjusted to deliver 0.1 units/mg ofFibrinogen or 25 Units/ml thrombin. Once prepared the thrombin wasplaced on ice until use. The temperature of the fibrinogen and thrombinprior to dispensing was 4° C.±2° C. Molds were removed from the -80° C.freezer and placed on a copper plate that was placed on top of dry ice.A repeat pipettor was filled with fibrinogen and second repeat pipettorwas filled with thrombin. Simultaneously 2 ml of fibrinogen and 300micro liters of thrombin were dispensed into each mold. Once the moldswere filled they were returned to the −80° C. freezer for at least twohours before being placed into the freeze dryer. Dressings were thenlyophilized as described above. Once complete the dressings were storedin low moisture transmission foil bags containing 5 grams of desiccant.

Trilayer dressings were manufactured as described previously¹, using thesame materials as described above.Subsequently, the dressings wereplaced under conditions of 100% relative humidity at 37° C. for varioustimes in order to increase their relative moisture content to desiredlevels. The dressings were evaluated visually and for their handling andother physical characteristics. Following this evaluation, a sample ofeach of the dressings was tested to determine their moisture content.The remaining dressings were performance tested in the EVPA, Adherenceand Weight Held assays.

Results

The results of the assays are given in the Tables below:

TABLE 1 Performance Data of Inventive Solid Dressings Exposure Time PeelTest Weight to 100% Humid- EVPA # Adherence Held (g) ity @ 37° C. %Pass/ (±Std. (mean ± (minutes) Moisture Total Dev.) Std. Dev.) 0 2.5 2/24.0 ± 0 148 ± 28.3 1 5.8 2/2   3.5 ± 0.71 123 ± 7.1  15 16 2/2  2.5 ±.71 108 ± 14.1 45 24 2/2 4.0 ± 0 168 ± 0   60 28 2/2 4.0 ± 0 273 ± 7.1 225 44 2/2  2 ± 0  58 ± 14.1 1200 52 ND ND ND

TABLE 2 Performance Data for Tri-layer Dressings Exposure Time to 100%Humid- EVPA # Weight ity @ 37° C. % Pass/ Peel Test Held (g) (minutes)Moisture Total Adherence (mean) 0 3 1/1 2.0 78 15 22 1/1 2.0 78 60 33.70/1 0.5 28

TABLE 3 Integrity and Handling Characteristics of Inventive SolidDressings Exposure Time During Hydration to 100% Humidity Prior ToHydration Force After @ 37° C. Surface Speed of Required for Hydration(minutes) Appearance Curling Integrity Flexible Hydration HydrationAppearance 0 Normal No Excellent No Normal No Normal (Smooth, No (Nocracks “skin”) or flaking off) 1 Normal ″ Excellent Yes ″ ″ ″ (Smooth,No (No cracks “skin”) or flaking off) 15 Normal ″ Excellent ″ ″ ″ ″(Smooth, No (No cracks “skin”) or flaking off) 45 Normal ″ Excellent ″ ″″ ″ (Smooth, No (No cracks “skin”) or flaking off) 60 Normal SlightExcellent ″ ″ ″ ″ (Smooth, No (No cracks “skin”) or flaking off) 225Normal Yes Excellent ″ ″ ″ ″ (Smooth, No (No cracks “skin”) or flakingoff) 1200 Normal Curling Excellent ″ n/d n/d Mottled & (Smooth, No Up on(No cracks lumpy “skin”) Itself or flaking off)

TABLE 4 Integrity and Handling Characteristics of Trilayer DressingsExposure Time During Hydration to 100% Humidity Prior To Hydration ForceAfter @ 37° C. Surface Speed of Required for Hydration (minutes)Appearance Curling Integrity Flexibility Hydration Hydration Appearance0 Normal No Good. Some No Normal No Normal delamination 15 Irregular NoGood. Some Yes Slow No Mottled delamination 60 Skinned Yes Good. SomeYes Very Slow Yes Very Mottled delamination and lumpy

Conclusions:

The monolithic dressings were fully functional at very high levels ofmoisture. As much as 28% moisture was found to retain completefunctionality When the moisture levels rose to 44%, the dressings werestill functional, however some of their activity was reduced Higherlevels of moisture may also retain some function. The originaldressings, at 2.5% moisture content, were not flexible, but had all theother desired properties including appearance, a flat surface,integrity, rapid and uncomplicated hydration and a smooth appearancepost hydration. Once the moisture content was increased to 5.8%, themonolithic dressings became flexible, while retaining theirfunctionality and desirable characteristics. They retained theirflexibility, without curling or losing their integrity or appearing toform excessive amounts of fibrin prior to hydration.

This contrasted with the tri-layer dressings, which began to lose theirdesirable characteristics upon the addition of moisture, and lost thementirely by the time moisture had increased to 33%.

Lyophilization Procedure

Frozen dressings were placed into a pre-cooled Genesis™ lyophylizer(Virtis, Gardiner, N.Y.). The chamber was sealed and the temperatureequilibrated. The chamber was then evacuated and the dressingslyophilized via a primary and secondary drying cycle.

EVPA Performance Testing

Equipment and Supplies:

-   In-line high pressure transducer(Ashcroft Duralife™ or equivalent)-   Peristaltic pump (Pharmacia Biotech™, Model P-1 or equivalent)-   Voltmeter (Craftsman™ Professional Model 82324 or equivalent)-   Computer equipped with software for recording pressure or voltage    information-   Tygon™ tubing (assorted sizes) with attachments-   Water bath (Baxter Durabath™ or equivalent), preset to 37° C.-   Incubation chamber (VWR™, Model 1400G or equivalent), preset to 37°    C.-   Thermometer to monitor temperatures of both water bath and oven-   Assorted forceps, hemostats, and scissors-   10 cc. and 20 cc. syringes with an approximately 0.6 cm hole drilled    in center and smaller hole drilled through both syringe and plunger.    This hole, drilled into the end of the syringe, will be used to keep    the plunger drawn back and stationary.-   O-rings (size 10 and 13)-   Plastic Shields to fit the 10 cc and 20 cc syringes (approximately    3.5 cm in length)-   P-1000 Pipetman™ with tips-   Sphygmomanometer with neonatal size cuff and bladder-   Programmable Logic Controller (PLC) to control the pumps to maintain    the desired pressure profile (Optional. Manual control may be used    if desired.)

1. Materials and Chemicals

-   Porcine descending aortas (Pel-Freez Biologicals™, Catalog #59402-2    or equivalent)-   Cyanoacrylate glue (Vetbond™, 3M or equivalent)-   18-gauge needle(s)-   0.9% Saline, maintained at 37° C.-   Red food coloring-   Vascular Punch(es), 2.8 mM or other-   Plastic Wrap

2. Artery Cleaning and Storage

-   1. Store arteries at −20° C. until used.-   2. Thaw arteries at 37° C. in H₂O bath.-   3. Clean fat and connective tissue from exterior surface of artery.-   4. Cut the arteries into ˜5 cm segments.-   5. The arteries may be refrozen to −20° C. and stored until use.

3. Artery Preparation for Assay

-   1. Turn the artery inside-out so that the smooth, interior wall is    facing outwards.-   2. Stretch a size 13 O-ring over a 20 cc syringe or a size 10 O-ring    over a 10 cc syringe with an approximately 0.6 cm (0.25 in) hole    drilled into one side.-   3. Pull the artery onto the syringe, taking care not to tear the    artery or have a too loose fit.

The artery should fit snugly to the syringe. Slide another O-ring of thesame size onto the bottom of the syringe

-   4. Carefully pull both O-rings over the ends of the artery. The    distance between the O-rings should be at least 3.5 cm-   5. Using the blade of some surgical scissors, gently scrape the    surface of the artery in order to roughen the surface of the artery.-   6. Use a 18-gauge needle to poke a hole through the artery over the    site of the hole in the syringe barrel (see note above)-   7. The tip of the biopsy punch is inserted through the hole in the    artery. Depress the punch's plunger to make an open hole in the    artery. Repeat a couple of times to ensure that the hole is open and    free of connective tissue.-   8. Patch holes left by collateral arteries. Generally this is done    by cutting a patch from a latex glove and gluing it over the hole    with cyanoacrylate glue. Allow the glue to cure for at least 10    minutes.-   9. Place the artery in the warmed, moistened container and place in    the incubation chamber. Allow the arteries to warm for at least 30    minutes.

4. Solution and Equipment Preparation

-   1. Check to see that the water bath and incubation chamber are    maintained at 29-33° C.-   2. Make sure that there is sufficient 0.9% saline in the pump's    reservoir for completion of the day's assays. Add more if needed.-   3. Place 0.9% saline and 0.9% saline with a few drops of red food    coloring added into containers in a water bath so that the solutions    will be warmed prior to performing the assay.-   4. Prepare the container for warming the arteries in the incubation    chamber by lining with KimWipes™ and adding a small amount of water    to keep the arteries moist.-   5. Check the tubing for air bubbles. If bubbles exist, turn on the    pump and allow the 0.9% saline to flow until all bubbles are    removed.

5. Application of the Dressing

-   1. Open the haemostatic dressing pouch and remove haemostatic    dressing-   2. Place the haemostatic dressing, mesh backing side UP, over the    hole in the artery-   3. Slowly wet the haemostatic dressing with an amount of saline    appropriate for the article being tested    -   NOTE: A standard (13-15 mg/cm² of fibrinogen) 2.4×2.4 cm        haemostatic dressing should be wet with 800 μl of saline or        other blood substitute. The amount of saline used can be        adjusted depending on the requirements of the particular        experiment being performed; however, any changes should be noted        on the data collection forms.    -   NOTE: Wet the haemostatic dressing drop wise with 0.9% saline        warmed to 29-33° C. or other blood substitute, taking care to        keep the saline from running off the edges. Any obvious        differences in wetting characteristics from the positive control        should be noted on data collection forms.-   4. Place the shield gently onto the haemostatic dressing, taking    care that it lies flat between the O-rings. Press lightly to secure    in place-   5. Wrap the artery and haemostatic dressing with plastic wrap-   6. Wrap with blood pressure cuff, taking care that the bladder is    adjacent to the haemostatic dressing.-   7. Pump up the bladder to 100-120 mMHg, and monitor the pressure and    pump again if it falls below 100 mMHg. Maintain pressure for 5    minutes.    -   NOTE: Time and pressure can be altered according to the        requirements of the experiment; changes from the standard        conditions should be noted on the data collection forms.-   8. After polymerization, carefully unwrap the artery and note the    condition of the haemostatic dressing. Any variation from the    positive control should be noted on the data collection form.

EXCLUSION CRITERION: The mesh backing must remain over the hole in theartery. If it has shifted during the polymerization and does notcompletely cover the hole the haemostatic dressing must be excluded.

Testing Procedure

6. Diagram of Testing Equipment Set-Up

A diagram of the testing equipment set-up is shown in FIG. 2. Someadditional components, not shown, may be utilized to read out (pressuregauge) or control the pressure within the system as desired.

7. Equipment and Artery Assembly

Fill the artery and syringe with red 0.9% saline warmed to 37° C.,taking care to minimize the amount of air bubbles within the syringe &artery. Filling the artery with the opening uppermost can assist withthis. Attach the artery and syringe to the testing apparatus, makingsure that there are as few air bubbles in the tubing as possible. Theperistaltic pump should be calibrated so that it delivers approximately3 ml/min. If available, the PLC should be operated according to apre-determined range of pressures and hold times as appropriate for thearticle being tested. If under manual control, the pressure/time profileto be followed is attained by manually turning the pump on and off whilereferencing the system pressure as read out by one or morepressure-reading components of the system. Following the conclusion oftesting, the haemostatic dressing is subjectively assessed with regardto adhesion to the artery and formation of a plug in the artery hole.Any variations from the positive control should be noted on the datacollection form.

Success Criteria

Haemostatic dressings that are able to withstand pressures for 3 minutesare considered to have passed the assay. When a haemostatic dressing hassuccessfully passed the assay the data collection should be stoppedimmediately so that the natural decrease in pressure that occurs in theartery once the test is ended isn't included on the graphs. Should theoperator fail to stop data collection, these points can be deleted fromthe data file to avoid confusing the natural pressure decay that occurspost-test with an actual dressing failure. The entire testing periodfrom application of the haemostatic dressing to completion must fallwithin pre-established criteria. The maximum pressure reached should berecorded on the data collection form.

-   -   NOTE: Typical challenge is 250 mMHg for three minutes in one        step, but that may be altered based on the article being tested.        Changes from the standard procedure should be noted on the data        collection forms.

Failure Criteria

Haemostatic dressings that start leaking saline at any point duringtesting are considered to have failed the assay.

-   -   NOTE: Build failures that are caused by artery swelling can be        ignored and the test continued or re-started (as long as the        total testing time doesn't fall beyond the established limit).

When leakage does occur, the pressure should be allowed to fall ˜20 mMHgbefore data collection is stopped so that the failure is easily observedon the graphs. The pressures at which leakage occurred should berecorded on the data collection form. Should the data collection stop inthe middle of the experiment due to equipment failure the data can becollected by hand at 5 second intervals until the end of the test orhaemostatic dressing failure, whichever happens first. The data pointsshould be recorded on the back of the data collection form, clearlylabeled, and entered by hand into the data tables.

Exclusion Criteria

If the total testing period exceeds the maximum allowed for thatprocedure, regardless of cause, results must be excluded. If there areleaks from collaterals that can't be fixed either by patching or fingerpressure the results must be excluded. If the test fails because ofleaks at the O-rings, the results must be excluded. If the mesh backingdoes not completely cover the hole in the artery, the results must beexcluded.

Adherence Performance Testing

8. Equipment and Supplies

Hemostat(s), Porcine artery and haemostatic dressing (usually aftercompletion of the EVPA Assay although it does not need to be performedto do the Adherence Assay).

9. Preparation of the Artery+Dressing

After application of the dressing without completion of the EVPA Assay,the dressing is ready for the Adherence Assay and Weight Limit Test (ifapplicable). After application of the dressing and subsequent EVPAAnalysis, the artery and syringe system is then disconnected slowly fromthe pump so that solution does not spray everywhere. The warmed, redsaline solution from the EVPA Assay remains in the syringe until theAdherence Assay and Weight Limit Test (if applicable) is completed.

Performance of the Adherence Assay

1. After preparation of the artery and dressing (with or without EVPAanalysis), gently lift the corner of the mesh and attach a hemostat ofknown mass to the corner.

-   -   NOTE: If the FD developed a channel leak during the performance        of the EVPA Assay, test the adherence on the opposite of the        haemostatic dressing to obtain a more accurate assessment of the        overall adherence.

2. Gently let go of the hemostat, taking care not to allow the hemostatto drop or twist. Turn the syringe so that the hemostat is near the topand allow the hemostat to peel back the dressing as far as the dressingwill permit. This usually occurs within 10 seconds. After the hemostathas stopped peeling back the dressing, rate the adherence of the bandageaccording to the following scale:

Dressing Performance Score Amount of Adherence 4  90+% 3 75-90% 2 50-75%1  ~50% 0.5 Only the plug holds the hemostat 0 No adherence

Exclusion Criteria

The mesh backing must remain over the hole in the artery. If it hasshifted during the polymerization and does not completely cover the holethe haemostatic dressing must be excluded.

Success Criteria

Dressings that are given an adherence score of 3 are considered to havepassed the assay.

Failure Criteria

If a dressing does not adhere to the artery after application and/orprior to performing the EVPA assay, it is given a score of 0 and failsthe adherence test. If a dressing receives a score ≦2, the dressing isconsidered to have failed the Adherence Assay.

Weight Held Performance Assay

After the initial scoring of the “Adherence Test”, weights may then beadded to the hemostat in an incremental manner until the mesh backing ispulled entirely off of the artery. The maximum weight that the dressingholds is then recorded as a measure of the amount of weight the dressingcould hold attached to the artery.

Moisture Assay

Moisture determinations were carried out using a Brinkman MetrohmMoisture Analyzer System. The system contains the following individualcomponents, 774 Oven Sample Processor, 774SC Controller, 836 Titrando, 5ml and 50 ml 800 Dosino Units and a 801 Stirrer. The system wasconnected to a computer using the Brinkman Tiamo software for datacollection, analysis and storage. The moisture system is set-up and runaccording to the manufactures recommendations and specifications tomeasure the moisture content of lyophilized samples using the KarlFischer method.

All components were turned on and allowed to reach operating temperatureprior to use. Lactose and water were run as standards and to calibratethe instrument. Once the machine was successfully calibrated, sampleswere prepared as follows. Dressing pieces weighing at least 30 mg wereplaced into vials and capped. The vials were placed in the 774 OvenSample Processor in numerical order, and one empty capped vial is placedin the conditioning space. The machine was then run to determine themoisture content (residual moisture) in the controls and samples.

1. A solid dressing for treating wounded tissue in a mammal comprisingat least one haemostatic layer conisisting essentially of thrombin and afibrinogen component, wherein said thrombin is present an amount betweenabout 0.250 Units/mg of fibrinogen component and 0.062 Units/mg offibrinogen component.
 2. The solid dressing of claim 1, furthercomprising at least one support layer.
 3. The solid dressing of claim 2,wherein said support layer comprises a hacking material.
 4. The soliddressing of claim 2, wherein said support layer comprises an internalsupport material.
 5. The solid dressing of claim 2, wherein said supportlayer comprises a resorbable material.
 6. The solid dressing of claim 2,wherein said support layer comprises a non-resorbable material. 7.(canceled)
 8. The solid dressing of claim 3, further comprising at leastphysiologically acceptable adhesive between said haemostatic layer andsaid backing layer. 9-11. (canceled)
 12. The solid dressing of claim 1,wherein said haemostatic layer also contains a fibrin crosslinker and/or a source of calcium ions.
 13. The solid dressing of claim 1, whereinsaid haemostatic layer also contains one or more of the following: atleast one filler, at least one solubilizing agent, at least one foamingagent and at least one release agent. 14-17. (canceled)
 18. The soliddressing of claim 1, wherein said haemostatic layer also contains atleast one therapeutic supplement selected from the group consisting ofantibiotics, anticoagulants, steroids, cardiovascular drugs, growthfactors, antibodies (poly and mono), chemoattractants, anesthetics,antiproliferatives/antitumor agents, antivirals, cytokines, colonystimulating factors, antifungals, antiparasitics, antiinflammatories,antiseptics, hormones, vitamins, glycoproteins, fibronectin, peptides,proteins, carbohydrates, proteoglycans, antiangiogenins, antigens,nucleotides, lipids, liposomes, fibrinolysis inhibitors and gene therapyreagents.
 19. The solid dressing of claim 1, wherein said haemostaticlayer is made from a single aqueous solution containing a mixture ofsaid fibrinogen component and said thrombin.
 20. The solid dressing ofclaim 1, wherein said haemostatic layer is cast as a single piece. 21.The solid dressing of claim 1, wherein said haemostatic layer issubstantially homogeneous throughout.
 22. The solid dressing of claim 1,wherein said haemostatic layer is composed of a plurality of particles,each of said particles consisting essentially of fibrinogen andthrombin.
 23. The solid dressing of claim 22, wherein said haemostaticlayer further contains at least one binding agent in an amount effectiveto improve the adherence of said particles to one another. 24.(canceled)
 25. The solid dressing of claim 1, wherein said haemostaticlayer is a monolith. 26-28. (canceled)
 29. The solid dressing of claim1, Wherein said fibrinogen component is a mammalian fibrinogen. 30-33.(canceled)
 34. The solid dressing of claim 29, wherein said mammalianfibrinogen is present in an amount between 1.5 mg/cm2 of thewound-facing surface of said dressing and 13.0 mg/cm2 of the woundfacingsurface of said dressing. 35-38. (canceled)
 39. The solid dressing ofclaim 19, wherein said therapeutic supplement is present an amount equalto or greater than its solubility limit in fibrin.
 40. A method oftreating wounded tissue in a mammal, comprising placing a solid dressingof claim 1 to said wounded tissue and applying sufficient pressure tosaid dressing for a sufficient time for enough fibrin to form to reducethe loss of blood and/ or other fluid from said wounded tissue.